Recombinant anti-epidermal growth factor receptor antibody compositions

Abstract

The invention relates to the field of recombinant antibodies for use in human cancer therapy. More specifically the invention provides compositions or mixtures of antibodies capable of binding human EGFR. Antibody compositions with 3 or more antibodies showed synergy in reduction of proliferation of representative cancer cell lines. Advantageous results have also been obtained with a composition comprising two different chimeric anti-hEGFR antibodies which show a new mechanism of action based on rapid and efficient receptor internalisation, induction of terminal differentiation and subsequent tumour eradication in an animal model. The antibodies of the invention can be manufactured in one bioreactor as a polyclonal antibody.

1. An antibody composition comprising at least two distinct anti-EGFR antibodies, wherein at least one of said anti-EGFR antibodies is selected from:
 (a) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 40) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 72) of 992, 
 (b) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 41) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 73) of 1024, 
 (c) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 42) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 74) of 1030, 
 (d) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 43) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 75) of 1042, 
 (e) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 44) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 76) of 1208, 
 (f) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 45) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 77) of 1229, 
 (g) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 46) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 78) of 1254, 
 (h) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 47) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 79) of 1257, 
 (i) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 48) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 80) of 1260, 
 (j) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 49) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 81) of 1261, 
 (k) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 50) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 82) of 1277, 
 (l) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 51) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 83) of 1284, 
 (m) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 52) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 84) of 1308, 
 (n) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 53) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 85) of 1320, 
 (o) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 54) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 86) of 1344, 
 (p) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 55) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 87) of 1347, and 
 (q) mixtures of two or more of said antibodies in (a)-(p). 
 
     
     
       2. The antibody composition of  claim 1 , wherein at least one of said anti-EGFR antibodies is selected from
 (a) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 40) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 72) of 992, 
 (b) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 42) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 74) of 1030, 
 (c) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 41) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 73) of 1024, 
 (d) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 55) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 87) of 1347, 
 (e) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 50) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 82) of 1277, 
 (f) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 46) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 78) of 1254, 
 (g) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 53) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 85) of 1320, 
 (h) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 48) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 80) of 1260, 
 (i) an antibody comprising CDR1CDR2 and CDR3 of the heavy chain (SEQ ID NO: 49) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 81) of 1261, 
 (j) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 51) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 83) of 1284, and 
 (k) mixtures of two or more of said antibodies in (a)-(j). 
 
     
     
       3. The antibody composition of  claim 1 , wherein said composition is selected from:
 (a) a composition comprising an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 40) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 72) of 992 and an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 42) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 74) of 1030, 
 (b) a composition comprising an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 40) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 72) of 992 and an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 41) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 73) of 1024, 
 (c) a composition comprising an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 40) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 72) of 992 and an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 43) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 75) of 1042, 
 (d) a composition comprising an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 40) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 72) of 992 and an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 53) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 85) of 1320, and 
 (e) a composition comprising an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 50) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 82) of 1277 and an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 41) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 73) of 1024. 
 
     
     
       4. An antibody composition comprising at least 2 distinct anti-human EGFR antibodies:
 (a) wherein a first distinct anti-EGFR antibody is an antibody capable of inhibiting the binding of and which binds the same epitope as an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 40) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 72) of antibody 992 to human EGFR; and 
 (b) wherein a second distinct anti-EGFR antibody is an antibody capable of inhibiting the binding of and which binds the same epitope as an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 41) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 73) of antibody 1024 to human EGFR. 
 
     
     
       5. The composition of  claim 4 , wherein:
 (a) said first distinct anti-EGFR antibody is an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 40) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 72) of antibody 99; and 
 (b) said second distinct anti-EGFR antibody is an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 41) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 73) of antibody 1024. 
 
     
     
       6. The composition of  claim 4 , wherein
 (a) said first distinct anti-EGFR antibody is an antibody comprising the V L  (amino acids 3-109 of SEQ ID NO: 72) and V H  (amino acids 3-124 of SEQ ID NO: 40) of antibody 992; and 
 (b) said second distinct anti-EGFR antibody is an antibody comprising the V L  (amino acids 3-114 of SEQ ID NO: 73) and V H  (amino acids 3-120 of SEQ ID NO: 41) of antibody 1024. 
 
     
     
       7. The composition of  claim 4 , wherein the first and second anti-EGFR antibodies do not inhibit the binding of each other to human EGFR. 
     
     
       8. The composition of  claim 4 , wherein at least one of the distinct anti-EGFR antibodies is capable of increasing the maximum binding capacity of the other distinct anti-EGFR antibody with respect to human EGFR. 
     
     
       9. The composition of  claim 4 , wherein the proportion of the first antibody relative to the second antibody in the composition is between 5% and 95% (mole/mole). 
     
     
       10. The composition of  claim 4 , wherein the first and second antibodies are of isotype IgG1 or IgG2. 
     
     
       11. The composition of  claim 4 , wherein the antibody capable of inhibiting the binding of and which binds to the same epitope as an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 40) and CDR1, CDR2, and CDR3 of the light chain (SE0 ID NO: 72) of antibody 992 is selected from antibody cluster 992 consisting of antibodies 1209, 1204, 992, 996, 1033, and 1220 in Table 12. 
     
     
       12. The composition of  claim 4 , wherein the antibody capable of inhibiting the binding of and which binds to the same epitope as an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 41) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 73) of antibody 1024 is selected from antibody cluster 1024 consisting of antibodies 1031, 1036, 1042, 984, 1024, 1210, 1217, 1221, and 1218 in Table 12. 
     
     
       13. The composition of  claim 4 , wherein the antibody capable of inhibiting the binding of and which binds the same epitope as an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 40) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 72) of antibody 992 to human EGFR is selected from:
 (a) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 44) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 76) of 1208, 
 (b) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 46) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 78) of 1254, and 
 (c) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 50) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 82) of 1277. 
 
     
     
       14. The composition of  claim 4 , wherein the antibody capable of inhibiting the binding of and which binds the same epitope as an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 41) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 73) of antibody 1024 is selected from:
 (a) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 43) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 75) of 1042 and 
 (b) an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 53) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 85) of 1320. 
 
     
     
       15. The composition of  claim 4 , wherein the composition does not contain further anti-EGFR antibodies in addition to said first and second antibodies. 
     
     
       16. The composition of  claim 4 , wherein the distinct antibodies are prepared for simultaneous, successive or separate administration. 
     
     
       17. The composition of  claim 4 , wherein the composition is capable of enhancing internalisation of EGFR. 
     
     
       18. The composition of  claim 4 , wherein the composition is capable of enhancing regression of A431NS tumours in vivo. 
     
     
       19. The composition of  claim 4 , wherein the composition is capable of inducing terminal differentiation in A431NS cells in vivo. 
     
     
       20. The composition of  claim 4 , wherein the composition is capable of up-regulating tumour involucrin expression in vivo. 
     
     
       21. A bi-specific binding molecule having the binding specificities of the antibody composition of  claim 4 . 
     
     
       22. The bi-specific binding molecule of  claim 21 , comprising the CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 40) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 72) of 992 and the CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 41) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 73) of 1024. 
     
     
       23. The bi-specific binding molecule of  claim 21 , comprising a dual-variable-domain antibody. 
     
     
       24. The bi-specific binding molecule of  claim 21 , comprising a bi-specific Fab-fragment or a bi-specific scFV. 
     
     
       25. A method of reducing EGFR signalling comprising administering to a composition of cells expressing EGFR an antibody composition of  claim 1 , or the bi-specific binding molecule of  claim 21 , thereby reducing the EGFR signalling. 
     
     
       26. A method of killing cells expressing EGFR comprising administering to a composition of cells expressing EGFR an antibody composition of  claim 1  or  4 , or the bi-specific binding molecule of  claim 21 , thereby killing the cells expressing EGFR. 
     
     
       27. A method of inducing apoptosis in cells expressing EGFR, comprising administering to a composition of cells expressing EGFR an antibody composition of  claim 1  or  4 , or the bi-specific binding molecule of  claim 21 , thereby inducing apoptosis. 
     
     
       28. A method of inhibiting proliferation of cells expressing EGFR comprising administering to a composition of cells expressing EGFR an antibody composition of  claim 1  or  4 , or the bi-specific binding molecule of  claim 21 , thereby inhibiting proliferation. 
     
     
       29. A method of inducing differentiation of tumour cells in vivo, comprising administering to an individual inflicted with cancer an antibody composition of  claim 1  or  4 , or the bi-specific binding molecule of  claim 21 , thereby inducing differentiation of the tumour cells. 
     
     
       30. The method of  claim 29 , wherein said differentiation is terminal. 
     
     
       31. The method of  claim 29 , wherein said differentiation is accompanied by an increase in involucrin expression. 
     
     
       32. A method for inducing internalisation of EGFR, comprising administering to cells expressing EGFR an effective amount of an antibody composition of  claim 1  or  4 , or the bi-specific binding molecule of  claim 21 , thereby inducing internalisation of EGFR. 
     
     
       33. A pharmaceutical composition comprising a combination of two or more antibodies of the composition of  claim 1  for simultaneous, separate or successive administration in cancer therapy. 
     
     
       34. The pharmaceutical composition of  claim 33 , further comprising at least one compound capable of inducing differentiation of cancer cells. 
     
     
       35. The pharmaceutical composition of  claim 34 , wherein the compound is selected from the group consisting of retinoic acid, phenylbutyrate, all-trans-retinoic acid, and active form vitamin D. 
     
     
       36. The pharmaceutical composition of  claim 33 , further comprising at least one chemotherapeutic or at least one additional antineoplastic compound. 
     
     
       37. The pharmaceutical composition of  claim 36 , wherein the chemotherapeutic compound is selected from the group consisting of adriamycin, cisplatin, taxol, doxorubicin, topotecan, fluoropyrimidine, oxaliplatin, and irinotecan. 
     
     
       38. A pharmaceutical composition comprising a combination of two or more antibodies of the composition of  claim 4  for simultaneous, separate or successive administration in cancer therapy. 
     
     
       39. The pharmaceutical composition of  claim 38 , further comprising at least one compound capable of inducing differentiation of cancer cells. 
     
     
       40. The pharmaceutical composition of  claim 39 , wherein the compound is selected from the group consisting of retinoic acid, phenylbutyrate, all-trans-retinoic acid, and active form vitamin D. 
     
     
       41. The pharmaceutical composition of  claim 38 , further comprising at least one chemotherapeutic or at least one additional antineoplastic compound. 
     
     
       42. The pharmaceutical composition of  claim 41 , wherein the chemotherapeutic compound is selected from the group consisting of adriamycin, cisplatin, taxol, doxorubicin, topotecan, fluoropyrimidine, oxaliplatin, and irinotecan. 
     
     
       43. A pharmaceutical composition comprising the bi-specific binding molecule of  claim 21  for simultaneous, separate or successive administration in cancer therapy. 
     
     
       44. The pharmaceutical composition of  claim 43 , further comprising at least one compound capable of inducing differentiation of cancer cells. 
     
     
       45. The pharmaceutical composition of  claim 44 , wherein the compound is selected from the group consisting of retinoic acid, phenylbutyrate, all-trans-retinoic acid, and active form vitamin D. 
     
     
       46. The pharmaceutical composition of  claim 43 , further comprising at least one chemotherapeutic or at least one additional antineoplastic compound. 
     
     
       47. The pharmaceutical composition of  claim 46 , wherein the chemotherapeutic compound is selected from the group consisting of adriamycin, cisplatin, taxol, doxorubicin, topotecan, fluoropyrimidine, oxaliplatin, and irinotecan. 
     
     
       48. The composition of  claim 4 , further comprising a third distinct anti-EGFR antibody, wherein said third distinct anti-EGFR antibody is an antibody capable of inhibiting the binding of and which binds the same epitope as an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 42) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 74) of antibody 1030 to human EGFR. 
     
     
       49. The composition of  claim 4 , further comprising a third distinct anti-EGFR antibody, wherein said third distinct anti-EGFR antibody is an antibody comprising the V L  (amino acids 3 to 114 of SEQ ID NO: 74) and V H  (amino acids 3-120 of SEQ ID NO: 42) sequences of antibody 1030. 
     
     
       50. The composition of  claim 4 , further comprising a third distinct anti-EGFR antibody, wherein said third distinct anti-EGFR antibody is an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 42) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 74) of antibody 1030. 
     
     
       51. The composition of  claim 48 , wherein said third distinct anti-EGFR antibody results in an enhanced binding to human EGFR of said first and/or second antibody. 
     
     
       52. The composition of  claim 48 , wherein said antibody capable of binding to the same epitope as an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 42) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 74) of antibody 1030 is selected from antibody cluster 1030 comprising antibodies 1195, 1030, 1034, 1194, 980, 981, 1246, or 1223 in Table 12. 
     
     
       53. The composition of  claim 48 , wherein the composition does not contain further anti-EGFR antibodies in addition to said first, second, and third antibodies.